FEATURE ARTICLE CUTTING EDGE Building bridges between cellular and molecular structural biology Abstract The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures. DOI: 10.7554/eLife.25835.001 ARDAN PATWARDHAN*, ROBERT BRANDT, SARAH J BUTCHER, LUCY COLLINSON, DAVID GAULT, KAY GRU¨ NEWALD, COREY HECKSEL†, JUHA T HUISKONEN, ANDRII IUDIN, MARTIN L JONES, PAUL K KORIR, ABRAHAM J KOSTER, INGVAR LAGERSTEDT‡, CATHERINE L LAWSON, DAVID MASTRONARDE, MATTHEW MCCORMICK, HELEN PARKINSON, PETER B ROSENTHAL, STEPHAN SAALFELD, HELEN R SAIBIL, SIRARAT SARNTIVIJAI, IRENE SOLANES VALERO§, SRIRAM SUBRAMANIAM, JASON R SWEDLOW, ILINCA TUDOSE, MARTYN WINN AND * *For correspondence: ardan@ GERARD J KLEYWEGT ebi.ac.uk (AP); [email protected] (GJK) Present address: †Electron Bio- Introduction can be used to link to other bioinformatics Imaging Centre, Diamond Light To obtain an integrated view of how molecular Source Ltd, Didcot, United resources such as the Universal Protein Resource Kingdom; ‡Computational machinery operates inside cells, biologists are (UniProt; uniprot.org; UniProt Consortium, Chemistry and Cheminformatics, increasingly combining structural data at differ- 2013). However, atomic models are not always Lilly UK, Windlesham, United ent length scales, obtained using a range of available for a variety of reasons, such as when Kingdom; §University of Vic - techniques such as electron tomography, elec- molecular averaging fails to obtain high-resolu- Central University of Catalonia, tron microscopy, NMR spectroscopy and X-ray tion features or the inherently lower resolution Barcelona, Spain crystallography. Structural data is held in public associated with molecules being imaged in more archives such as the Electron Microscopy Data complex or even cellular environments. In such Reviewing editor: Werner Bank (EMDB; emdb-empiar.org; Tagari et al., cases, the identification of features often relies Ku¨ hlbrandt, Max Planck Institute 2002), the Electron Microscopy Public Image on prior knowledge or correlation of structural of Biophysics, Germany Archive (EMPIAR; empiar.org; Iudin et al., data obtained at different scales. This is an open-access article, 2016), and the Protein Data Bank (PDB; wwpdb. Once features have been identified, segmen- free of all copyright, and may be org; Bernstein et al., 1977) tation (defined here as the decomposition of the freely reproduced, distributed, Integration between PDB and EMDB data is 3D volume into regions that can be associated transmitted, modified, built based on atomic models in the PDB that have with defined objects) can be employed to facili- upon, or otherwise used by anyone for any lawful purpose. been fitted to or built into EMDB volume maps. tate and visualise the interpretation of the map. The work is made available under For purified biological molecules or larger For example, in a recent study the segmentation the Creative Commons CC0 defined complexes this approach is done rou- of electron and soft X-ray tomography recon- public domain dedication. tinely. Sequence information from the models structions was used to study leakage and Patwardhan et al. eLife 2017;6:e25835. DOI: 10.7554/eLife.25835 1 of 11 Feature article Cutting edge Building bridges between cellular and molecular structural biology Figure 1. Segmentation of Plasmodium falciparum–infected erythrocytes. Soft X-ray tomography shows loss of mechanical integrity of the red cell membrane in the final stages of egress. Panels A-C depict schizonts treated with a selective malarial cGMP-dependent protein kinase G inhibitor (C2), and panels D-F depict schizonts treated with a broad-spectrum cysteine protease inhibitor, E64, which allows parasitophorous vacuole membrane (PVM) rupture but prevents erythrocyte membrane rupture, resulting in merozoites trapped in the blood cell. (A) Slice from tomogram of C2-arrested schizont. (B) Outlines of erythrocyte membrane (red), PVM (yellow), and parasites (cyan) in the tomogram slice in A.(C) 3D rendering of the schizont. The vacuole (yellow) is densely packed with merozoites (cyan) that have been collectively rather than individually rendered, for clarity. The overall height of the cell is ~5 mm. (D) Tomogram slice from an E64-arrested schizont, shown with outlining of membranes in E. Remnants of the PVM are visible. (F) 3D rendering of the schizont. Figure and legend adapted with permission from Hale et al. (2017). Scale bar 1 mm. DOI: 10.7554/eLife.25835.002 breakage of the membranes in erythrocytes higher resolution afforded by sub-tomogram infected by Plasmodium falciparum, and docu- averaging provides more structural detail, dis- mented the dramatic changes in the morphology playing sub-tomogram averages at the original of cells during egress (Hale et al., 2017). The soft tomogram positions and orientations may reveal X-ray tomograms provided overviews of the important information about the organization membrane compartments in intact, vitrified cells and distribution of the object within a cellular (Figure 1). It should be noted that the word ’seg- and functional context. If properly annotated mentation’ may have different interpretations: for such data can be further mined with other ques- example, in whole animal, pre-clinical and medi- tions in mind by other researchers. For example, cal imaging, segmentation includes a concept of researchers recently created composite maps of a model that is used for fitting of the features. In Lassa virus particles by inserting the sub-tomo- this manuscript we limit the definition to the sep- gram average structure of the Lassa virus glyco- aration of density into distinct sub-domains. protein spike back into the original tomographic In tomography, where multiple copies of reconstructions, revealing the organisation and nearly identical objects are found, 3D sub-tomo- copy number of the spikes on the virus surface gram averaging and 3D classification may be (Figure 2; Li et al., 2016). Another example employed to obtain higher resolution recon- revealed the lateral clustering of viral membrane structions. This process often involves combining proteins mediating membrane fusion information from multiple tomograms. Since the (Maurer et al., 2013). Patwardhan et al. eLife 2017;6:e25835. DOI: 10.7554/eLife.25835 2 of 11 Feature article Cutting edge Building bridges between cellular and molecular structural biology Figure 2. Arrangement of Lassa virus glycoprotein spikes on the virion surface. Left to right: A slice from a tomographic volume of Lassa viruses, a sub-tomogram average of the glycoprotein spike, and the sub-tomogram average inserted back onto a virus reconstruction. Images adapted from Li et al., 2016 (under a CC BY 4.0 license). DOI: 10.7554/eLife.25835.003 The archiving of segmentation data in EMDB transformation data, the Protein Data Bank in entries was identified as an area requiring urgent Europe (PDBe) organised an expert workshop on attention in previous workshops on “Data-Man- “3D Segmentations and Transformations - Build- agement Challenges in 3D Electron Microscopy” ing Bridges between Cellular and Molecular in 2011 (Patwardhan et al., 2012) and “A 3D Structural Biology” in December 2015. The Cellular Context for the Macromolecular World” objectives were: in 2012 (Patwardhan et al., 2014), as was the . To identify data models and formats for improved biological annotation of structural data representing segmentation and transfor- to make it more accessible to the wider biological mation data that could provide support audience and to enable integration with struc- for structured biological annotation, thus tural and other bioinformatics resources. Crucially facilitating their use by EMDB and for data integration we need “structured biologi- enabling data-exchange between different cal annotation” which is here defined as the asso- software packages . To gain a better understanding of the ciation of data with identifiers (e.g., accession challenges involved in the annotation of codes from UniProt) and ontologies taken from electron microscopy data and develop well established bioinformatics resources. (Ontol- requirements in terms of tools and strate- ogies are formal collections of statements defin- gies to facilitate annotation. ing concepts, relationships and constraints; for example, the mitochondrial large and small ribo- Here we report and discuss the main out- comes of the workshop, which was attended by a somal subunits are parts of the mitochondrial ribosome which, in turn, is a part of the mitochon- range of participants including software develop- drion). To our knowledge, none of the segmenta- ers, users of segmentation software, ontology tion formats widely used in electron microscopy experts, and experts in structure and data and related fields currently support structured archiving. biological annotation. Furthermore, spatial trans- formations relating sub-tomograms to their par- ent tomograms are not currently captured in Data models and file formats for EMDB. Moreover, wider usage